SUMMARY: The Preclinical Development and Clinical Monitoring Facility (PDCMF) of the Experimental Transplantation and Immunology Branch supports the development and implementation of new protocols involving adoptive immune cell therapies through preclinical development, translational implementation of clinical products and preservation and analysis of patient blood and tissues during clinical trials. The work of this core is supported by close collaborative relationships with the Cell Processing Service of DTM, for support of microarray analysis and development of clinical products; the ETIB Flow Cytometry Facility, for support of sorting of clinical products for research endpoints; and the laboratory of Ronald Gress, for technical support in RNA and DNA isolation and quantitative assays. Several novel protocols involving adoptive transfer of T cells have been implemented in recent years as a result of this process. (1) In ETIB protocol 11-C-0016 (P.I. Daniel Fowler), patients with multiple myeloma have received an autologous hematopoietic stem cell transplant followed by infusion of a distinctive autologous T cell product, T1.Rapa cells, in a phase one trial of escalating T1.Rapa cell doses; through expanding the scale of product manufacturing, serial infusions of T1.Rapa cells were then implemented following pentostatin induced lymphodepletion. The T1.Rapa cells were generated through expansion of host T cells by CD3/CD28 bead costimulation in the presence of IFN-alpha and rapamycin, to generate a persistent cell product with Th1/Tc1 activity. We provided translational scale-up of the T1.Rapa product developed in Dr. Fowler's laboratory, provided operating protocols and materials documentation in support of the FDA submission, and characterized changes induced by the T1.Rapa culture using microarray, flow cytometry and cytokine production assays. These demonstrated skewing toward Th1/Tc1 central memory cells with elevated expression of both IFN-induced and autophagy-associated genes. In the ongoing clinical trial, we assessed serial changes in effector and regulatory T cell populations in blood and in bone marrow (the main tumor site), and demonstrated concurrent changes in gene expression and T cell phenotype consistent with increased Th1/Tc1 activity following T1.Rapa infusion. Finally, using gDNA-based T cell receptor sequencing (Immunoseq by Adaptive Biotechnologies) we identified novel clonal expansions following infusion and tracked their persistence in patients with prolonged remission. (2) In collaboration with Dr. Fowler, we have assessed the effects of use of the pentostatin/cytoxan regimen he developed for lymphodepletion in support of a mesothelioma protocol using a partially humanized anti-mesothelin antibody-immunotoxin developed by Dr. Pastan's laboratory. Development of resistance to immune therapies can be linked to the immunogenicity of the infused antibody immunotoxin agent. This year we collaborated with Dr. Pastan's laboratory to investigate regulation of immunogenicity of immunotoxins by HLA-DR/DP/DQ expression (Mazor et al, AAPSJ, 2017) (3) We have supported the implementation by Dr. James Kochenderfer of Chimeric Antigen Receptor (CAR) therapy to treat lymphoma, leukemia and myelomas. In the first trial of the use of donor-derived anti-CD19 Chimeric Antigen Receptor (CAR) T cells in patients with relapsed or persistent lymphoma following allogeneic transplant (Protocol 10-C-0054: P.I. James Kochenderfer (ETIB)), PDCMF staff in Cell Processing Section optimized the CAR transduction and subsequent CAR T cell expansion process, developed the operational protocols and documentation for IND submission and trained the clinical staff in this new procedure. Subsequently PDCMF staff supported the implementation of a new lentiviral human-immunoglobulin-based anti-CD19 CAR vector that Dr. Kochenderfer had designed, which entered into clinical trials this past year (16-C-0054). In the PDCMF laboratory we have supported processing and storage of apheresis products for further research as well as daily patient biospecimens to track the spike of immune response engendered by CAR therapy. Using multi-parameter flow cytometry, we tracked the presence of CAR+ T cells following adoptive transfer and have demonstrated the expansion of anti-CD19 CAR+ T cells in the blood concurrent with the onset of anti-tumor activity, approximately one week after adoptive transfer (Kochenderfer et al, Blood 2013). When the 10-C-0054 protocol was expanded to treat follicular lymphoma and acute lymphoblastic leukemia, we tracked anti-CD19 CAR+ T cells in peripheral blood in these patients (Brudno et al, J Clin Oncol, 2016). When Dr. Kochenderfer developed a new CAR construct directed against the BCMA receptor expressed on myeloma cells (Carpenter et al, Clin Cancer Res, 2013), PDCMF staff in DTM validated the procedures for transduction of the final GMP grade construct and prepared the SOP for generation of the expanded CAR product. In the PDCMF laboratory, we tracked anti-BCMA CAR+ T cells by flow cytometry, demonstrating a massive expansion in blood and marrow, concurrent with depletion of myeloma cells (Abbas et al, Blood, 2016). PDCMF has also supported biospecimen processing for Dr. Kochenderfer's involvement in a multicenter CRADA trial of another anti-BCMA CAR product (16-C-0025). Finally, in the past year, PDCMF staff in DTM have validated clinical-grade generation of a new anti CD30 CAR developed by Dr. Kochenderfer (17-C-0048); this new construct opens disorders such as Hodgkin's lymphoma to CAR therapy trials. (4) Dr. Luca Gattinoni (ETIB) has demonstrated that persistent CD8+ memory stem cells (Tscm) are the most effective T cells for adoptive immune therapy against tumors. To support utilization of Tscm as the base cell for CAR transduction and adoptive immune therapy, PDCMF staff in Cell Processing have validated the process of isolation of naive CD8 cells, conversion of these cells into Tscm and expansion into a clinical product, and have developed Standard Operating Procedures (SOP), Protocol Specific Instructions (PSI) and Chemistry, Manufacturing and Controls (CMC) section of IND submissions to the FDA. This protocol, implemented as 10-C-0054M, will shortly be used to assess the effectiveness of a Tscm cellular base for antiCD19 CAR therapy. (5) Dr. Christian Hinrichs has initiated therapeutic trials for treatment of viral-induced tumors, specifically the respiratory and genital associated tumors induced by human papilloma virus (Protocols 16-C-0154, 16-C-0160, 17-C-0016). These studies have involved introduction of checkpoint inhibitors of the PD-1/PD-L1 system, and adoptive immunotherapy utilizing expanded T cells transduced to express a transgenic T cell receptor directed against either the E6 or the E7 viral protein. PDCMF staff in Cell Processing has assisted in the optimization of protocols and development of documentation for FDA approval of the transgenic T cell receptor cells. The PDCMF laboratory has supported processing and storage of blood and apheresis products, used both to monitor response and to provide a research collection for further cellular analyses. Furthermore the PDCMF staff has supported the development of procedures for processing epithelial tumor biopsies, collected prior to and after treatment. These biopsies have to be divided and appropriately stored for RNA-based analyses, immunohistochemistry in paraffin and frozen sections, and separation into single cell suspensions to establish cultures of tumor cells and of infiltrating tumor-reactive T cells. This processing is carried out in collaboration with the ETIB T Cell Facility and with Dr. Hinrichs' researchers.